IE47150B1 - Process for the preparation of antibacterial agents - Google Patents

Abstract

3-N-formyl derivatives of 3'',6'-di-N-acyl-kanamycin A and 2',3'',6'-tri-N-acyl-kanamycin B, process for their preparation, and their use as intermediates for production of antibacterially active 1-N-(substituted) derivatives of kanamycins A and B by alkylation and subsequent removal of the formyl and acyl groups.

Description

This invention relates to a novel process for the preparation of aminoglycoside antibiotics and with novel intermediates for use in such process, and is particularly concerned with a process for the preparation of 1-N- alkyl-substitutedkanamycin derivatives and with selectively N-protected kanamycin der vatives as intermediates for use in the process.

Examples of such 1-N-alkyl-substituted kanamycin derivatives are described in _ Patent Specification Number 42452 ; others are described in Belgian Patent Number 855,709.

In order to prepare such compounds from the readily available fermentation product kanamycin, it is desirable to protect all the amino groups other than the 1-amino group.

Alkylation may then be effected preferentially on the amino group on the 1-position and isolation of the final l-N-substituted product is thereby simplified. It is an object of this present invention to provide a process fox the preparation of l-Nsubstituted kanamycin derivatives by providing such selectively N-protected intermediates.

Thus, according to the invention there is provided a process for preparing.compounds of the formula: - 2 47150 where R is an amino or hydroxyl group and R is a lower alkyl group, any carbon atom of which, other than the carbon atom attached to the nitrogen atom, may optionally be substituted with an hydroxyl or an amino group; which process comprises alkylation of a compound of the formula: where R is a formyl group, R 4 or a benzoyl group and R is to produce a compound of the is a C2 a hydroxy formula: to alkanoyl group group or a group NHR ; (III) 4 wherein R to R are as previously defined; and wherein any amino group present in R1 may optionally be protected; removal of the groups 3 1 R , R and any amino-protecting group in R ; and isolation of the compound of formula (I). - 3 471S0 In this specification the term lower alkyl indicates that such a group contains from 1 to 6 carbon atoms and may be straight or branched chain. Particular examples of substituted-lower alkyl groups are th/s] -4-amino-2-hydroxy butyl and 1,3-dihydroxyprop-2-yl groups· The blocking group is preferably an acetyl group.

This process for the preparation of compounds of formula (I) comprises as an initial step alkylation of a compound 1 of formula (II) in order to introduce the substituent R onto the amino group at the 1- position. Such a reaction may be performed in a number of ways well known to those skilled in the art. Fcr example, alkylation may be achieved by reductive alkylation using an appropriate aldehyde or ketone, or a compound such as described in Belgian Patent Number 851,777.

The second step of the process comprises removal of the blocking groups r\ from the 2'-amino group, if present, and the 6' and 3-amino groups and also the formyl group on the 3-amino group. In some instances where the 1-N-substituent itself bears an amino substituent group it may be desirable to protect this group during the course of the process and it will then be necessary to remove this amino-blocking group as well in the final step of the process. There are various conditions for completely removing araino-blocking groups, well known to those skilled in the art, and they will naturally depend on the nature of the protecting group employed and the environment of the protected amine. The medium employed may be anhydrous or agueous and in particular instances it may be acidic or basic to various strengths. - 4 η 47150 For example the benzyl group, when present, can be removed by catalytic hydrogenolysis in a conventional manner in the presence of a palladium catalyst. The alkanoyl or benzoyl blocking groups may generally be removed by hydrolysis under mild basic conditions, for example by treatment with dilute sodium hydroxide at 65°C for several hours. These conditions also remove the formyl group. The product (I) may finally be purified, if desired, by conventional techniques, for example, by crystallisation or by chromatography.

The process is exemplified by the preparation of l-N-^(S)4-amino-2-hydroxybutylJkanamycin A. In this case the protected 4 kanamycin intermediate of formula (II) wherein R is a hydroxyl group and R3 is an acetyl group is alkylated by a reductive alkylation using an appropriate aldehyde or a compound as described in Belgian Patent Number 851,777. Thus, 3-benzyl-6-(S)-dihydroxyraethyltetrahydro-1,3-oxazin -2-one may be used and the reductive alkylation may be performed using sodium borohydride or preferably sodium cyano(or other compound) borohydride as the reducing agent. Excess aldehyde' and reducing agent may be used to ensure complete reaction of the free 1-amino group. Suitable solvents are aqueous methanol or aqueous dioxan; dimethylformamide or dimethylsulphoxide are also suitable. An acid e.g. acetic acid may also be added with advantage. The reaction is generally performed at room temperature and is usually complete within 5 or 6 hours. The crude product may be isolated by evaporation of the solvent and may be further purified, if desired by recrystallisation or chromatography. Generally, however, it will be more convenient to use the crude product directly in the next stage of the process. - 5 47150 Thus the crude product is dissolved in dilute sodium hydroxide and heated at 65°C for a period of about 5 hours to effect removal of the formyl and acetyl blocking groups. After neutralisation, the product, preferably separated from the inorganic material, is subjected to a conventional catalytic hydrogenation to remove the benzyl protecting group. A period of 4 or 5 hours at 60°c and a pressure of 60 p.s.i. is generally sufficient to effect complete removal of the benzyl group and the product is finally worked up in a conventional manner and purified if necessary by ion exchange chromatography to give the final product. Since there is only ona reactive amino group in the starting material of formula (II) the final product consists substantially of the required l-N-substituted isomer and the difficult separation of closely related isomers is thereby avoided.

The reaction may also be performed in an exactly analogous manner using 2'^(e'-tri-N-acetyl-S-formyl-kanamycin B to provide the corresponding l-N-substituted kanamycin B derivative.

The alkylation reaction may also be performed using a 20 hydroxy-substituted aldehyde or ketone to give 1-N-hydroxyalkyl substituted kanamycin derivatives such as are described in • Belgian Patent Number 855,709. Thus, for example, when 1,3-dihydroxy acetone is used in the process the product of formula (I) is obtained 'wherein R^ is a l,3-dihydroxyprop-2-yl group. - 6 4 7150 The compounds of formula (II) are themselves novel compounds according to the invention. They may be prepared from 3,6'-di-N-acyl-kanamycin A or 2',3,6'-tri-N-acyl-kanamycin B by a reaction which involves first formylation of the remaining unsubstituted 1 and 3-amino groups and secondly a selective hydrolysis step which has been found, surprisingly, to effect the removal of the formyl group from the 1-amino position while leaving the formyl group on the 3-position untouched.

The preparation of suitable acyl-kanamycin derivatives substituted on the 3 and the 6' amino groups in kanamycin A and additionally on the 2'-amino group in kanamycin B with a C2 to alkanoyl group or a benzoyl group is described in Belgian Patent Number 853,564 . For example, the preparation of 3,6'-di-N-acetyl-kanamycin A is described via a selective 0 to N acetyl migration reaction. Other derivatives may be prepared in a similar manner.

The formylation reaction may be performed in a number of different ways, well known to those skilled in the art. For example, formylation may be achieved using a mixture of formic and acetic anhydrides but we have found that the reaction may conveniently be performed using 4-nitrophenylformate. Suitable solvents for the reaction are aqueous dioxan, aqueous tetrahydrofuran or dimethylformamide, and the reaction is performed by adding excess (e.g. a three fold excess) of 4-nitrophenylformate, in portions, to the kanamycin derivative. - 7 47150 A base is preferably added to aid the reaction and an organic base, e.g. triethylamine may be employed for this purpose. At least one equivalent of base is preferably added for each reactive amino group. The formylation reaction is generally complete within 24 hours at room temperature and the product may then be isolated by conventional techniques, e.g. by evaporation of the solvent or by precipitation and may be further purified if desired.

The selective hydrolysis stage is performed with the 1,3-di-N-formyl product dissolved in water or in an aqueous organic solvent, e.g. aqueous dioxan or aqueous tetrahydrofuran and the pH of the solution is carefully adjusted to within the range 12.0 - 12.5 with an alkali, e.g. by adding dilute sodium or potassium hydroxide. The solution is stirred at room temperature for several days, the course of the reaction being followed by thin-layer chromatography, until conversion to the desired 3-Nformyl derivative of formula (II) is complete. The solution is then neutralised by addition of acid, or more conveniently by adding an ion-exchange resin in the hydrogen-ion form. This has the advantage of preventing a build up of inorganic material in solution and has the added advantage that any by-product in which both the 1 and'3-formyl groups have been cleaved is adsorbed onto the resin. The product is finally isolated by conventional techniques, e.g. by evaporation to a low volume and precipitation with an organic solvent, e.g. isopropanol. - 8 47150 Further purification if desired may be achieved by chromatography but in general the product is sufficiently pure for use in the preparation of compounds of the formula (I) directly.

The process for the preparation of compounds of the formula (II) may be performed using 3,6'-di-N-acetyl kanamycin A to give 4 the compound of formula (II) wherein R is acetyl ard R is a hydroxy group. Similarly 2', 3”, 6’-tri-N-acetyl-kanamycin B may be used to 4 3 3 give the compound of formula (II) wherein R is a group NHR and R is acetyl.

The invention is illustrated by the following Examples in which Examples l’and 3 are examples of preparation of novel intermediates of the formula (II) ard Examples 2 and 4 are examples of the novel process for preparation of compounds of the formula (I).

Thin layer chromatography was performed on silica plates using the solvent system stated. The spots were visualised after drying the plates by spraying with a 5% solution of t-butyl-hypochlorite in cyclohexane, drying the plates at 100°C for 10 minutes in a ventilated oven, cooling and spraying with starch-potassium iodide solution.

Thin layer electrophoresis was performed on 20 cm silica plates with a potential difference of 900 volts applied for 45 minutes.

The electrolyte was a mixture of formic and acetic acids giving a pH value of 2, detection was performed as above.

Amberlite and Sephadex are Trade Marks. - 9 47150 EXAMPLE 1 Preparation of 3,6'-di-N-acetyl-3-N-formyl-kanamycin A (A) 4-Nitrophenylformate (6.88 g) was added portionwise over 45 minutes to a suspension of 3,6'-di-N-acetyl-kanamycin A (3.9 g) in water (21 ml) and tetrahydrofuran (21 ml) containing triethylamine (6.91 g). The reaction was stirred at room temperature overnight and then evaporated to low volume replacing the final traces of water by azeotropic distillation with isopropanoi (final volume 35 ml). 3,6'-Di-N-acetyl-1, 3-di-N-formyl-kanamycin A (2.41 g) precipitated from the solution and was filtered and dried in vacuum at 50°C.

On thin layer electrophoresis (pH = 2) the compound gave a valuecf 0.09 with respect to kanamycin A. On t.l.c. using methanol, ethylacetate, ammonia, water (40:40:1:30) as eluant it gave Rf 0.42. The proton n.m.r. spectrum showed signals at = 7.96 p.p.m. (formyl proton), 8.02 p.p.m. (formyl proton) and 2.02 p.p.m. (6 acetyl protons).

(B) The product from (A)(2.4 g) was dissolved in water (72 ml) and the pH of the solution was adjusted to 12.0 12.5 with 2N sodium hydroxide solution (2.0 ml) . The solution was stirred at room temperature for 7 days maintaining the pH between 12.0 and 12.5 and was then neutralised by the addition of Amberlite IR120 ion exchange resin (H+ form) (10ml). The resin was filtered and the filtrate was concentrated to low volume, the final traces of water being removed by displacement with dimethylformamide (5 ml) at reflux. - 10 - 471 Isopropanol (30 ml) was then added slowly to the dimethylformamide solution and the precipitate of 3',6'-di-N-acetyl-3-N-formyl kanamycin A (1.75 g) was filtered and dried in vacuum at 40°c.

The compound was pure enough for use in the next stage 5 of the process. A sample was further purified by chromatography on CM Sephadex C25 (NH^ + form) using O.lM ammonium hydroxide solution as eluant. It gave a C-13 n.m.r. spectrum in full agreement with the required structure and which confirmed mono formylation of the 3-amino group. Thin layer electrophoresis (pH = 2) gave a value of 0.43 with respect to kanamycin A.

On t.l.c. using methanol, ethylacetate, ammonia, water (40:40: 1:30) as eluant the compound gave an Rf value of EXAMPLE 2 Preparation of l-N-^(S)-4-amino-2-hydroxybutylj-kanamycin A Sodium cyanoborohydride.(0.21 g) in water (40 ml) was added to 3,6'-di-N-acetyl-3-N-formyl-kanamycin A(2.0 g), 3-benzyl-6-(S)- dihydroxymethyl -tetrahydro-2H-l,3-oxazin-2-one (2.38 g) and copper sulphate (0.1 g) in water (66 ml) and methanol (322 ml) over 5 hours. The reaction mixture was evaporated to dryness under reduced pressure and the residue was treated with water· (25 ml) and methylene chloride (25 ml). The aqueous phase was evaporated to dryness and the residue was heated with 3N sodium hydroxide solution (20 ml) at 65-70°C for 4 hours.

The solution was cooled and neutralised with concentrated hydrochloric acid using good fume extraction (HCN given off) and passed down a 600 ml column of Amberlite CG-50 ion exchange resin (NHj + form) eluting the inorganics with water, and the amino5 glycoside materials with 0.2N ammonium hydroxide solution.

The aminoglycoside fractions were evaporated to dryness and the residue was dissolved in a mixture of methanol (13 ml), acetic acid (13 ml) and water (13 ml) and hydrogenated over 5% palladium on charcoal (0.4 g) at 60°C and 60 p.s.i. for 4 hours.

The catalyst was filtered and the filtrate was evaporated to dryness. The residue was dissolved in water (20 ml) and chromatographed on a 200 ml CG-50 (NH^ + form) ion exchange column using ammonium hydr«ttte solution (0.25 - 1.0M) as eluant. Evaporation of the appropriate fractions gave 1-N-pS)-4-amino-2-hydroxybutyl] kanamycin A (0.75 g, 37%) identical on thin layer chromatography and thin layer electrophoresis with an authentic sample.

EXAMPLE 3 Preparation of 2',3,6'-tri-N-acetyl-3-N-formyl-kanamycin B (A) Benzyl chloroformate (128 g) was added over a period of minutes to a solution of kanamycin B sulphate (80.5 g) and sodium carbonate (76 g) in water (400 ml), and the solution was stirred for 1 hour at room temperature. The precipitate was collected by filtration, washed with water, with dilute hydrochloric acid and with water and dried to yield penta-N-benzyloxycarbonyl-kanamycin B (yield 125.7 g) .

(B) Penta-N-benzyloxycarbonyl-kanamycin B (230.8 g) was added portionwise over 15 minutes to a stirred solution of acetic anhydride (189 ml) in pyridine (346 ml) and dichloromethane (346 ml) and the suspension was stirred at room temperature for 48 hours. The solution was poured into a mixture of dichloromethane (1.5 litres) and water (2.3 litres). The organic phase was separated and washed with dilute hydrochloric acid (pH 4) and with water. The solvent was evaporated to a volume of 0.75 litre and the solution poured into diethyl ether (4 litres). The precipitate of tetra-O-acetyl-penta-N-benzyloxycarbonyl-kanamycin B was collected by filtration and dried under vacuum (yield 244.8 g, 92.6%).

(C) A solution of tetra-O-acetyl-penta-N-benzyloxycarbonylkanamycin B (33.0 g) in tetrahydrofuran (132 ml), water (66 ml) and acetic acid (3.3 ml) was hydrogenated over 5% palladium on charcoal catalyst (3.3 g) at 50°C and 50 p.s.i. for 7 hours. The catalyst was removed by filtration and the filtrate was concentrated to a volume of 25 ml. The residue was treated with ammonium hydroxide (7N, 81.5 ml) and stirred overnight at room temperature. The solution was evaporated to dryness and the residue chromatographed on a column 4715® of Amberlite CG-50 ion-exchange resin in the ammonium ion form (2 litres) eluting with water and then with 0.01 aqueous ammonia.

Fractions containing the desired product were combined and evaporated to yield 2',3,S'-tri-N-acetyl-kanamycin B (9.37 g, 61.6%), m.p. 191 - 198°C. (decomp.) M (KBr) 1650, 1550 cm-1, Rf 0.11 (6:3:1: * maxK. methanol, diethyl ether, water, 0,880 aqueous ammonia), Rf 0.11 (40:40:30:1 methanol, ethyl acetate, water, 0.880 aqueous ammonia).

(D) p-Nitrophenylformate (153.1 g) was added portionwise over minutes to a cooled solution of 2’,3,6’-tri-N-acetyl-kanamycin B 10 (93 g) in a mixture of water (465 ml) tetrahydrofuran (558 ml) and triethylamine (154 g). The reaction mixture was stirred at room temperature overnight and then concentrated to a low volume under reduced pressure. The aqueous concentrate was diluted with isopropanol to precipitate the product, the residual water being renoved by azeotropic distillation with isopropanol. The product was finally collected by filtration, washed with isopropanol and dried under vacuum to yield 2',3,6'-tri-N~acetyl-l,3-di-N-formyl kanamycin B (89.2 g) m.p. 310 - 318°C (decomp.)}) (KBr) 1665, 1545 cm 1, Rf 0,49 (6:3:1:2 methanol, diethyl ether, water, 0.880 aqueous ammonia), 20 Rf 0.54 (40:40:30:1 methanol, ethyl acetate, water, 0.880 aqueous ammonia). - 14 47150 (F) A solution of 2' ,3, 6'-tri-N-acetyl-l, 3-di-N-formyl kanamycin B (86.9 g) in water (2.61 litres) was taken to pHl2 to 12.5 with ION sodium hydroxide solution. The reaction was kept at room temperature for 4 days readjusting the pH of the solution to 12 with aqueous sodium hydroxide as necessary, and the solution was then neutralised by the ·{· addition of Amberlite IR 120 resin (H form). The mixture was filtered and the filtrate was chromatographed on Amberlite 200 ion-exchange resin (ammonium-ion form, 5.5 litres) eluting with water and then with 0.IN aqueous ammonium hydroxide. Evaporation of the appropriate fractions gave 2’,3, 6'-trl-N-acetyl-3-N-formyl kanamycin B (55.4 g), m.p. 266 268° (decomp.) V (KBr) 1665, 1555 cm 1, Rf 0.49 (40:40:30:1 methanol, - max. ethyl acetate, water 0.880 aqueous ammonia) . The structure was confirmed by C-13 n.m.r. spectroscopy.

EXAMPLE 4 Preparation of l-N-(l,3-Dlhydroxyprop-2-yl) kanamycin B A solution of 2',3,6'-tri-N-acetyl-3-N-formyl kanamycin B (8.8 g) in methanol (880 ml) and water (176 ml) was treated with 1,3-dihydroxyacetone (4.0 g) and sodium cyanoborohydride (3.9 g) and the pH of the resulting solution adjusted to 6.5 with 5N hydrochloric acid. The solution was heated under reflux for 18 hours, cooled and further 1,3-dihydroxyacetone (4.0 g) aid sodium cyanoborohydride (3.9 g) were added. The pH was re-adjusted to 5.5 with hydrochloric acid and the solution was heated under reflux for a further 6 hours. The methanol was removed under reduced pressure and the aqueous concentrate was 25 chromatographed on a column of Amberlite 200 ion-exchange resin in the ammonium ion form eluting with water. - 15 47150 Fractions containg the required 2' ,3, 6'-tri~N-acetyl-3-N-formyl-lH- (1,3-dihydroxyprop-2-yl) kanamycin B were combined and concentrated under reduced pressure to a volume of 40 ml.

. The concentrate was treated with ION aqueous sodium hydroxide (40 ml) and the mixture was heated on a steam bath for 16 hours. The cooled solution was neutralised with concentrated hydrochloric acid, water added to a volume of 1 litre and the mixture chromatographed on a column of Amberlite 200 ion-exchange resin (ammonium-ion form, 3 litres). The column was washed with water to ranove inorganics and then with IN agueous ammonia to elute the aminoglycoside materials.

The crude product was re-chromatographed on Amberlite CG-SO ion-exchange resin (ammonium-ion form, 2 litres) eluting with water, 0.05N aqueous ammonium hydroxide and finally 0.1N aqueous ammonium hydroxide. Evaporation of appropriate fractions gave 1-N-(1,3-dihydroxyprop-2-yl) kanamycin B (3.2 g) identical on thin layer chromatography with a reference sample.

Claims (24)

CLAIMS:

1. of the formula: A process for preparing compounds (I) where R is an amino or hydroxyl group and R is a lower alkyl group, any carbon atom of which, other than the carbon atom attached to the nitrogen atom, may optionally be substituted with an hydroxyl or an amino group; which process comprises alkylation of a compound of the formula: (II) 2. 3 where R is a formyl group, R is a to alkanoyl group or a 4 -3 benzoyl group and R is a hydroxy group or a group NHR ; to produce a compound of the formula: (III) 1 4 wherein R to R are as previously defined, and wherein any amino group 1 2 present in R may optionally be protected; removal of the groups R R^.and any amino-protecting group in rS and isolation of the compound of formula. (I).

2. A process as claimed in claim 1 wherein R^ is an S -4araino-2-hydroxybutyl group.

3. A process as claimed in claim 1 wherein R 1 is a 1,3dihydroxyprop-2-yl group.

4. A process as claimed in any preceding claim wherein R^ is an acetyl group.

5. A process as claimed in claim 4 wherein the compound of formula (II) is 3, 6'-di-N-acetyl-3-N-formyl-kanamycin A.

6. A process as claimed in claim 4 wherein the compound of formula (II) is 2', 3'',6'-tri-N-acetyl-3-N-formyl-kanamycin B.

7. A process as claimed in any preceding claim wherein the alkylation is achieved by reductive alkylation. - 18 47150

8. A process as claimed in claim 7 wherein the reductive alkylation is achieved with 3-benzyl-6-(S)-dihydroxymethyl-tetrahydro-1,3-oxazin2-one, and the benzyl protecting group is removed by catalytic hydrogenation after removal of R 2 and r\

9. A process as claimed in claim 7 wherein the reductive 5 alkylation is achieved with 1,3-dihydroxyacetone.

10. A process as claimed in claims 7 to 9 wherein the reducing agent is sodium borohydride or sodium cyanoborohydride.

11. A process as claimed in claim 1 substantially as hereinbefore described with reference to Examples 2 and 4. 10

12. A compound of the formula: 2 3 wherein R is a formyl group, R is a to C& alkanoyl group or a 4 3 benzoyl group and R is a hydroxy group or a group NHR .

13. A compound as claimed in claim 12 wherein R^ is an acetyl 15 group.

14. 3,6*-Di-N-acetyl-3-N-formyl-kanamycin A.

15. 2', 3,6'-Tri-N-acetyl-3-N-formyl-kanamycin B.

16. A process for preparing a compound of the formula (II) as defined in claim 12 which comprises formylation of a compound of the 2 3 4 formula (II) wherein R is hydrogen and R and R are as defined in claim 12 followed by selective hydrolysis in an aqueous or aqueous organic 5 solvent at a pH of 12.0 to 12.5 to remove the formyl group from the 1-amino position.

17. A process as claimed in claim 16 wherein is an acetyl group.

18. A process as claimed in claim 16 or 17 wherein the starting material of formula (II) wherein R is a hydrogen is 3, 6'-di-N-acetyl 10 -kanamycin A.

19. A process as claimed in claim 16 or 17 wherein the starting material of formula (II) wherein R is hydrogen is 2',3,6'-tri-Nacetyl kanamycin B.

20. A process as claimed in any one of claims 16 to 19 wherein 15 the formylation is achieved with 4-nitrophenylformate.

21. A process as claimed in claim 20 wherein the formylation is performed in aqueous dioxan, aqueous tetrahydrofuran or dimethylformamide in the presence of a base.

22. A process as claimed in any one of claims 16 to 21 wherein 20 the selective hydrolysis is performed with dilute sodium or potassium hydroxide.

23. A process as claimed in claim 16 substantially as hereinbefore described with reference to Examples 1 or 3.

24. -. A compound of the formula (II) as claimed in claim 12 whenever prepared by a process as claimed in any one of claims 16 to 23.